Characterization of a Fission Yeast Gene, Gpa2, That Encodes a Subunit Involved in the Monitoring of Nutrition

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Characterization of a fission yeast gene, gpa2, that encodes a subunit involved in the monitoring of nutrition

Takako Isshiki, Nobuyoshi Mochizuki, 1 Tatsuya Maeda, 2 and Masayuki Yamamoto s Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Tokyo 113, Japan

The Schizosaccharomyces pombe gpa2 gene was cloned by hybridization with a eDNA for Dictyostelium discoideum G,vl. It encodes a homolog of G-protein ot-subunits with 354 amino acids and a predicted molecular mass of 40,522. Disruption of gpa2 slows cell growth but is not lethal. Cells defective in gpa2 mate and sporulate readily in the presence of plentiful nutrition, bypassing the requirement of nitrogen starvation for the initiation of sexual development. These phenotypes mimic those of cells defective in cyrl encoding adenylyl cyclase. The level of cAMP in gpa2 null mutants is only one-third of the wild-type level. Mutations in gpa2 that are likely to inhibit the GTPase activity of the gene product cause a slight increase in intracellular cAMP levels and result in leaky sterility. The cAMP level reaches 20 times as high as the wild-type level if a cell carries both this type of gpa2 mutation and a null mutation in pdel encoding phosphodiesterase. Cells defective in gpa2 fail to produce cAMP in response to glucose stimulation. These results suggest that Gpa2 is involved in the determination of the cAMP level according to nutritional conditions, most likely as a positive regulator of adenylyl cyclase. [Key Words: Schizosaccharomyces pombe; G protein; cAMP production; glucose; sexual differentiation] Received August 6, 1992; revised version accepted October 12, 1992.

Cells of the fission yeast Schizosaccharomyces pombe receptors that carry seven membrane-spanning domains. initiate sexual development, namely mating, meiosis, The receptors on the cell membrane bind their specific and sporulation, in response to nitrogen starvation (Leu- ligands, and this signal is transmitted into the cell by the pold 1970; Egel 1973). Nitrogen starvation reduces the G proteins (Gilman 1987; Dohlman et al. 1991). Our pre- intracellular cAMP level by 50% (Fukui et al. 1986; Mo- vious screen identified the S. pombe gpal gene encoding chizuki and Yamamoto 1992). This reduction is a signal an a-subunit of a G protein that we concluded to be for induction of the stell gene, which encodes a tran- coupled with the mating pheromone receptors (Obara et scription factor responsible for the activation of genes al. 1991). We have now identified an S. pombe gene that involved in mating and meiosis (Watanabe et al. 1988; encodes another Get subunit and named it gpa2. Genetic Sugimoto et al. 1991 ). Carbon starvation also reduces the analysis of gpa2 suggests that this gene product is in- cAMP level, even more sharply than nitrogen starvation volved in the regulation of the intracellular cAMP level (N. Mochizuki and M. Yamamoto, unpubl.). However, in accordance with nutritional conditions. Its most prob- excessive carbon starvation apparently shuts down any able function is modulation of adenylyl cyclase activity. developmental activities. Partial carbon starvation can Thus, it is likely that a GoL-subunit plays a role in trans- augment the induction of sexual development by nitro- duction of a signal from nutritional conditions, although gen starvation. It is thus conceivable that fission yeast the identity of the signal has still not been determined. has a mechanism that detects the availability of nutri- ents and regulates the level of intracellular cAMP. How cells monitor the availability of nutrition is an Results interesting but poorly understood question. It now appears that our analysis of a G-protein homolog in S. Cloning of the gpa2 gene pombe has provided a clue to this question. S. pombe genomic DNA was analyzed by Southern blot- Heterotrimeric G proteins are generally coupled with ting using cDNAs encoding Dictyostelium discoideum Goal and Go~2 (Pupillo et al. 1989) as probes. A few restriction fragments hybridized to these probes under low Present addresses: 1National Institute for Basic Biology, Okazaki 444, stringency conditions (data not shown), and we cloned Japan; 2Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA. an 8.0-kb EcoRI fragment that gave a clear hybridization 3Corresponding author. band with the G~I cDNA. Subcloning and partial se-

GENES & DEVELOPMENT 6:2455-2462 91992 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/92 $3.00 2455 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press

Isshiki et al. quence analysis indicated that this fragment carried a -- 77:'7: :' ,r: ~7 :a;,~:~, :,V ;, ;,=,;,: ~V':':'V ;, :~:V:~ 7 ;,V:::: : :, .~T:x!V.~T;a.mVV,.~,;~::WWVV:,-'?,~::':Vr gene encoding a Got homolog, hereafter called gpa2. Be- :-" ;,at :,; 77.:7;,7: ; 7:7=, 7 :, r:T:7 vaq~';,.; 7A: ;: :~: :: ;,~ 7;:~.:,7/ "79; 7 A77 T :T,6,~,C;~ 7~::..-277T. ~. 4 2 ;,~,; "-T T .7.a~,~a 7 a3,aa=a,,=-'-a-TY;aaaT"7 : :ATCCava~A: 7 a'.;~*XT,:;;:;CT,~v,XZ:AAAzTT"TS:o(;TaTA;TTCA:;'2GA]Tq;A cause the 3' end of the gpa2 gene was not found on the 9- 7.:~A7 ,KAT:~T'7~.'Z ,r : ;APT 7;7:" 7 ;TAAT,~.7 ~Z~.7 ;A A : a 7D:TT: qA, '7"YA,~:: ATA ".'WTG :',X T .~,AZ~': :,;,~,~; :~ 9 : :: ; :~-~: :;, :~ ;V:::;':: :V :'-. V;, ;,V ::aVVT~V: :: V;* a ;7~C':':,:~7V;,a,~<~,X,XT;k:~;,...":VT~..;, EcoRI fragment, we cloned a 2.4-kb PstI fragment that : ~ v : F x covers the missing part of the gene. A restriction map of

::a : :-:-::~:~ = :~= := : :,;: ::-: 7: ::-v ;rv ::-v ;,;.::,::7~:;:a:: 7:;:. :,;.-: .:a: ~,a,~.~,,~: ,,:;~,=..~ :,:7"r;/,>~.~:,a;,~,= the gpa2 locus is shown in Figure 1, and the nucleotide - : : sequence of the entire gpa2 open reading frame (ORF) : :4 ::>_a ::;.: :;::: '- 7 a_-,.; 7=:77~ :~a.~; .:_a;, : : :':a: ,=.:.~: .a ~A~:a7: ~C'JV~7~.a77.:: 3T;_Ta~22;TTr:TZTA VCTTt: :4 ::'a rr: 7"7 :,:7:" : ; r t,:7 *Xa;:7;,~ 7CC';A7;, e, :77 77 7"~;77:,; ),(77CT'STq 57.3TqP'~ AT;TT3Ta~TfiAGTT;72 ~ and its flanking regions was determined (see Fig. 2). This 424 7,~ : 7 77a 777 : 7 ;77: aYA: .k .~:AT:'7:~.'T. ;7"77:;:: ::,:~; A,~.aTT"7"D 77'_: 777;77"7: TTI~,,:,;,YT ;T :A~T~L sequence suggested the existence of a 405-bp-long intron in gpa2, which was confirmed by cDNA cloning using the polymerase chain reaction (PCR)method (Saiki et al. 1988) (data not shown). Many mammalian Got genes have an intron at the same position as gpa2 (Kaziro et al. 1991). The gpa2 gene has a coding capacity of 354 amino acids. The deduced Gpa2 protein exhibits 39.3% amino ...; ~,::7:;-:77 777 :a a.~ : .:.~ :,7 TC :27 ~ 7.'-7Ca 7: ~,~7~7 ~..A;~TA.Ka,~7"7 7:;CT,~A~,A;TAT7~ :JTkTA7 acid identity with D. discoideum Gotl, whose cDNA was used as the probe. Gpa2 is 40.7% identical with D.

. :4 :a~ a A=*-k 7 7:: : : 7 a:~: : 7t,::. ,~:7C 7.a,X; : 7"7:::_~ ;,;,: 77 :CC7~ :.77 UT'77APTC:'TT"T:TTC'TKAT,~7 discoideum Got2, 40.1% with human Gi2ot, and only 2'24 7 7 a:TZ'Tk?:a ~ aT :7 7:'Tk:AkATaCqZZ; A.aT&:;~:=, A:::; T; ::.A=,:" : ;GaA'7 k : ;A2C:::7 T :T.a3,:;A~.A..~:;A: : 24 aS : 77:'7 a :7 : V .~ .:~ ;:C a a':a7"P : TkT*Tk,~,~T,:,=: : :::,X~,:a: :=CL;,A:S'V,~Va A;T7 Ta:?';:-AVa, Taa.AVk:X":*7; 36.2% with S. pombe Gpal. It is most similar to Sac- :-24 :" :~777"77; ::,=?a: =a:':'7 ;-77:',%k:7-:777,= 7:':;,&a ;aT" :. : . ;;,;-7; ~'!*77;,A;,;7"Ta~U;,aV;, :':':: :" :W :;.~, ,~r,~;V" :-.4 7V~a :7C : A<: 7= :: 7 a .7~7 a-7 aA .~: :aT 7 : 977": ;. ;&;, "7;,7-7;: Va: & 77:4,T TG 7.~:77;T;~ :; ;7 ;7a: charomyces cerevisiae Gpa2 (42.4%) among G-protein ot-subunits identified so far (Fig. 3). Figure 2. The nucleotide and deduced amino acid sequences of gpa2. Numbering starts at the presumed initiation codon. Un- derlined regions match the consensus sequence for splicing in S. Disruption of the gpa2 gene pombe (Russell 1989). The region between a KpnI site and a ScaI site is shown, which has been sequenced in both directions. The One of the two gpa2 alleles in a diploid strain, JY765, nucleotide sequence data reported here have been submitted to was disrupted, as described in Materials and methods. the DNA Data Bank of Japan, EMBL, and GenBank nucleotide The structure of the disrupted allele, in which nucle- sequence data bases under accession number D13366. otides -22 to + 1514 in Figure 2 were deleted and replaced by a ura4 + cassette, is schematically shown in Figure 1. Heterothallic haploid cells carrying the disrupted gpa2 allele were obtained by tetrad dissection of by genetic crosses. The homothallic derivatives mated the diploid strain. They were viable but were smaller in and sporulated readily in nitrogen-rich medium (Fig. 4B) cell size (Fig. 4E) and grew 1.4 times slower than the wild and grew very poorly. These phenotypes of the gpa2- type. Homothallic gpa2-disrupted cells were constructed disrupted cells were reminiscent of those of the cyrl- disrupted cells, which have no measurable activity of adenylyl cyclase and no measurable amount of cAMP (Maeda et al. 1990; Kawamukai et al. 1991). Addition of t t l lJ I ]; ,.I 2 mM cAMP and 5 mM caffeine to the medium, the latter of which is a phosphodiesterase inhibitor, could suppress pGPE1 3H I the observed phenotypes of the gpa2-disrupted cells: pGPR I Heterothallic cells resumed normal cell size and growth rate (Fig. 4F), and homothallic cells did not enter ectopic sexual development (Fig. 4C). These observations suggested that loss of function of gpa2 could cause a decrease in the level of intracellular cAMP, and measurement of the amount of cAMP in the cell demonstrated | I lkb that this was the case (Table 1, experiment 1). Figure 1. A restriction map and schematic illustration of disruption of the gpa2 gene. A pUC119-based plasmid, pGPE13H, carries a 4. 8-kb HindIII-EcoRI fragment. Using the EcoRI ter- Mutations affecting the GTPase activity of Gpa2 minal region of this insert (solid bar), a 2.4-kb PstI fragment was Mutations at either of the two amino acid residues in cloned in pUC119 and the resulting plasmid was named pGPR. mammalian Gsot, namely Arg-201 and Gln-227, are able A restriction map of the gpa2 locus was deduced from the in- to inhibit the GTPase activity of the protein and bring serts of these two plasmids and is shown at the top. The open oncogenic characteristics to it (Landis et al. 1989). Mu- arrow indicates the translated region of gpa2 interrupted by an intron. The structure of the linear fragment used to disrupt the tations in the corresponding positions of Gi2o~ also cause gpa2 gene is shown at the bottom. The hatched box represents a similar effect (Lyons et al. 1990). The two residues are an S. pombe ura4 + cassette. Restriction sites are abbreviated as conserved in Gpa2 as Arg-176 and Gln-202. We mutated follows: (B) BamHI; (E)EcoRI; (H) HindIII; (Hc)HincII;/K) KpnI; Arg-176 to His and Gln-202 to Leu according to the pro- (P) PstI; and (S) ScaI. cedure described by Kunkel {1985). These mutations

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S. pombe Ga monitoring nutrition

SpGpa2 1 XTIFNGLSR--SG .... gSK~LN---SKIEKQIENASKK ...... DKKIYKVLLLGAS 4 3 DdGRI 1 MGNICGK PHLGSP .... WE IKANQH- - -INS LLKQAR SK ...... LEGEIKLLLLGAG 45 ously (Mochizuki and Yamamoto 1992). More explicit DdG~2 I ~G- ICASS .... N .... {gGEKTNTD - - -ZNLSZEKERKK ...... KHNEVKLLLLGAG 40 SpGpa I 1 IG- - CMSSKYADTSGGEVI QKKLSDTQTSNSSTTGSQNA~ ( 33 aa ) + GGND IKVLLLGAG 84 evidence that R176H and Q202L mutations can cause ScGpa2 1 JG-LC~SSEKNGSTPDTQTASAGSDN--VGKAKVPPKQE. (83aa) +NDKELKVLLLQAG 132 hGi2~ 1 l~G - - CTVSA ...... IDKAAAER S KMI EKNLR E DGEK ...... AARE V~LLLLGAG 42 elevation of the intracellular cAMP level is shown be- A SpGpa2 44 DSGKSTISKQIKILNKMG~S0ERIMTFIPVZRRNLLESAKTLVKIZVQKGINLDPLGTHN ID3 low. DdG~I 46 ZS~KST~AK~MKI~HLNGFNDBIIKSSEKTZZYNWTVGSMRVLVNAAEELKZGISENNKEA 105 DdGU2 41 JSGKSTZSEQMKZXHQSGTSSIRRKEFKpIXTRJZLDNMIVLLDGMGRLGHTZDPSNSDA I00 Sp~pal 8 DSOKTTIMKQMRLLYSPGFS~VVRKQYRV~ZFENI~SSLCLLLEAMDMSNVSLLPENEKY 144 ScGpa2 133 JSGKSTVL(~LKI~HONGFSZOEIKEYIPLIYOJLLmIORNLIQARTRPMVNLEpECBLT 192 hGl2~ 43 ZS(~KSTIV~QMKIIHBDGTSESICRQYRAVVYSNTIQSIMAIVKAmGNLQXDFADPDSRA 102 Phenotypes of strains that carry either R176H SpGpa2 104 CEI~EKFNPTP ...... ~RLINANI~AITSLWSAHS--VRSC-TYGND-SVLIDSAP 151 or Q202L on the chromosome DdGGI 106 A---SRISNDLGDHPN~--~VLT-ARLA~D~KALWADPG--IQNTFQRSS|F-QLNDSAA 155 Dd~(~ i01 AVI~IKE~TSLQASIVTDCW~RL-NEDQ~KKIKAL~TDPG--VKQAM~RANEFSTLPDSAp i$7 SpGpal 145 RAVZLRKHTS~PNEP ...... FSPE~YEAVHASTLDTK+-LRTVQSCGTNLS-LLDNFY 193 ScGpa2 193 UQDLSRTMSYEMPNNYT--~Q-FPEDIAGVISTL~ALPSTQDLVNGPNASKF.YLMD~TP 248 Replacement of a chromosomal gpa2 allele by either the hGi2~ 103 DDA--RQLFALSCTAEEQ-~VL-PDDLSGVIRR~WADKG--VQACFGRSREY-QLNDSAA 154 R176H or the Q202L allele was performed using a gpa2- C S~Gg>a2 152 YPFSRADEIC~RHyVPTIDDZLRSRNSTL~ISEISFTLDHLQ-IRNFDVG~RT~RRKWZ 210 disrupted strain, JZ415 (h 9~ ade6-M216 leul ura4-D18 DdGul 156 ~YFDSIDRISQPL~LPSENDVLRSRTKTTGIIETVFEIQNST-FRg~DVGGQRSIRKKWM 214 DdG~2 158 YPFDSIDR~TSpVYZPTDODILMTRVMTRGVHETNFE~GKZK+PRLVDVG~QRSERKKWt. 216 SpGpal 194 ~YODMIDR~FDPQYIPSDQDILHCRIKTTG~SEETFLLNRHH-yRFFDVGGQRSRRRKWl 252 gpa2::ura4 + ), as a recipient. A 3.4-kb BamHI-SalI (PstI} ScGpa2 249 ~MENPTRZTSPHTR~TQQDZLRSRQMTS~FDTVID~GSDIKMH~DVOG~RS|RKKWI 30g hGi2~ 155 YYLNDLBRIAOSDYIPTQQDVLRTRVKTTGZVJTHFTFKDLH-FK~FDVOGQRSlRKKNI 213 fragment carrying either of the mutant alleles was intro-

G S~Gpa2 211 YCFEN~HSTX~CVSLNDYDKKLYERAAPER~RLVESZSLFDSIINSQWFMHSS~ILFL~ ~ 270 duced into the recipient. Ura- transformants, which ap- DdG~I 215 HCFQEVTAVIFC~ALSETDLKLYB--DDTTNRHQSSLKLFKE~CNTKWFANTAMILFLNK 272 Dd~(~2 217 SCFDDVTAVV~C~ALS~YDLLLYB--DNSTNRN~ESLRVFSDVC~-SWFVNTPIILpL~gK 273 parently had lost the disrupted allele, were selected us- S~Gpal 253 HCFSN~TALL~LVSLAGTDQCLVI--DNSGNQ~Q]EALLLWDS~CNSSNPSESA~ILFLNK 310 ScGpa2 309 HCPDNVTLVZPCVSLSEYDQTLMZ--DKNQNRF~SLVLPDNZVNSRNFARTSVVLFLiK 366 ing 5-fluoro-orotic acid (5-FOA). Successful replacement hGi2fx 214 HCPEGVTAZI~CVALSAYDLVLAE--DEEM~RMHESMKLFDSlCNNKWFTDTSIILFLIIK 271 of the gpa2 allele was confirmed by Southern blotting $~Gpa2 271 FDLFRKKLEBVPFQDY~PQYEOKN~VKSI ...... TRYZLWLFVNPSIN-R 314 DdG~I 273 RDI~SEKXTKTPITVCgKEED~p~TYEGC ...... 8EFIKOQFZNQ--S-E 314 (data not shown). The mutants thus obtained were D~G~2 274 SDLPRE~I~SVDLSETFPE~KGGRDYERA ...... 8N~fIKRRF-WQ-I~-~ 315 S~GDal 311 5DLPKR~GSH~PIQKM~PDYQEVG~TPTFVQTQCPLADNAVRSGMYYFYLKF-E.SLN. R 369 ScGpa2 367 ~DLFAEKLRKVpMENYFPDYT~SDINEA ...... AKYZLWRFV-Q-L~-R 408 crossed with appropriate strains, and Ura + derivatives, hGi2~ 272 KDLFEEKITHSPLTICFPEYT~ASKYDRA ...... ASYZOSKF-E-DLlgKR 314 namely JZ692 (h 9~ ade6-M216 leul gpa2 R176H) and JZ593 I S~Gpa 2 315 - AK H N~ y p HI TT~VDTSM T KVVFSAVK~T i LQ HSLK ~ AOM p 354 (h 9~ ade6-M216 leul gpa2Q2~ were constructed for DdGul 315 NPKKSIYPRLTCETDTNNZLVVFNAVKDIVLNLTLGEAOMIL 355 DdG~2 316 TEQKAIYSHITCATDTNNII%VVPEAVKDIIFTQCVMKAOLYS 357 S~Gpa 1 370 IAS R SCYCHFTTATDTSLLQRVMVSVQDT INS NNLQ S L -M p 408 further analysis. They were poor in mating, although ScG~a~ 409 +ANLSZYPHVTQ&TDTSNIRLVFAAIK~TXLENTLKDSGVL0 449 hGi 2(~ 315 KDT KEI TTHPTC~TD TK~Q I~V ~DAVTDVI ZK N ~LKDCG L p 35~ their sterility was leaky (Table 2); and they contained a slightly higher level of cAMP (Table 1, experiment 2). Figure 3. Comparison of the deduced amino acid sequence of S. pombe Gpa2 with D. discoideum Gcd and Ga2 (Pupillo et al. 1989), S. pombe Gpal {Obara et al. 1991), S. cerevisiae Gpa2 Combination of gpa2 mutations with disruption (Naka~uku et al. 1988), and human Gi2~ (Beals et al. 1987). of pde 1 Amino acid residues conserved in Gpa2 and at least two of the other G~ proteins are shaded. The regions indicated by A, C, G, The above results suggested that the Gpa2 protein either and I are supposed to interact with guanine nucleotides (Halli- stimulates the production of cAMP or inhibits its degra- day 1984; Dever et al. 1987). Conserved amino acid substitu- tions are grouped as follows: V, L, I, M; F, Y, W; K, R; D, E; Q, N; and S, T.

were termed R176H and Q202L. We then constructed three types of plasmids that express the wild type, the R176H allele, and the Q202L allele, respectively, from the adhl promoter, by inserting a 2.4-kb PstI fragment carrying each allele into the NdeI site of an expression vector, pART3 (Kelly et al. 1988). They were named pAGWT {wild type), pAGH (R176H), and pAGL (Q202L). A homothallic haploid strain, JY450, was transformed with these plasmids or the vector pART3. Compared with the control cells carrying pART3 (vector), cells transformed with pAGWT (gpa2 +) were less efficient in mating and sporulation (Fig. 5B). Cells transformed with either pAGH (gpa2 R176tt) or pAGL (gpa2 Qa~ were much less efficient (Fig. 5C, D). Thus, these transformants mimicked strains that have high levels of intracellular cAMP (Maeda et al. 1990; DeVoti et al. 1991; Figure 4. Morphology of gpa2-disrupted strains. JY450 (h 9~ Mochizuki and Yamamoto 1992). The cAMP level was wild type), JZ415 (h 9~ gpa2::ura4+), JY333 (h- wild type) and elevated in these transformants (Table 1, experiment 3); JZ393 (h - gpa2::ura4 +) were cultured freshly in MM. For JZ415 however, the elevation was not striking, although these and JZ393, the culture was made with and without the addition of 2 mM cAMP and 5 rnM caffeine. Phase-contrast micrographs transformants showed unambiguous phenotypes in mat- were taken. (A) JY450 (h 9~ wild type); (B) JZ393 (h 9~ ing. This probably reflects that the cAMP level is gpa2:: ura4 +) without cAMP and caffeine; (CI JY415 with cAMP strongly feedback regulated in fission yeast cells and that and caffeine; (D) JY333 (h- wild type}; (E) JZ393 (h- even a small increase or decrease in this level can signif- gpa2::ura4 +) without cAMP and caffeine; {F) JZ393 with cAMP icantly affect cell physiology, as was discussed previ- and caffeine. Bar, 10 ~m.

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Isshiki et al.

Table 1. cAMP level in various strains cAMP level Strain Relevant genotype Plasmid (pmole/mg of protein) Experiment 1 JY333 h - gpa2 + 3.6• JZ393 h - ~gpa2 1.3 _0.1 1.2 _0.1 Experiment 2 JY450 hg~ + 3.4_+0.1 JZ692 h9~ RI 76H 4.4_+ 0.1 JZ593 h g~gp a2 Q2~ 3.8 + 0.2 Experiment 3 JY450 hg~ + pART3 2.5 + 0.1 1.9 • JY450 hg~ + pAGWT(gpa2 +) 2.5 • 0.1 2.5 • JY450 hg~ + pAGH(gpa2 R176H) 4.0 • 0.3 2.9 • 0.3 JY450 hg~ + pAGL(gpa2 ~176 3.2 • 0.3 2.7 • 0.1 Experiment 4 JY450 hg~247 + 2.3 - 0.2 JZ666 hg~ 8.0 -+ 1.2 JZ655 h9~ 2.9 -+ 0.1 JZ656 3.2 -+ 0.1 JZ657 hg~ 96.6 - 4.3 JZ658 56.1 - 4.1 JZ659 h9~176 59.1 • 2.8 JZ660 52.7 • 12.1 Experiment 5 JZ666 hg~ 10.7 • 0.1 JZ667 14.5 • 0.3 JZ655 hg~ 2.6 • 0.4 JZ656 2.6 • 0.2 JZ657 h9~ 34.2 • 0.9 JZ658 53.4 -*-- 2.5 JZ659 h9~176176 64.9 - 0.7 JZ660 46.5 • 0.2 Cells grown in MM at 30~ were harvested at a concentration of 5 x 106 to 1 x 107 cells/ml and processed for cAMP assay. Each sample was assayed in duplicate. Two values are given for one strain when two independent samples were assayed.

dation. To differentiate between these two possibilities, mamoto 1992; Table 1, JZ666 and JZ667). If Gpa2 were a we combined R176H and Q202L mutations with a null negative regulator of this enzyme, the R176H or the mutation in pdel (Mochizuki and Yamamoto 1992), also Q202L mutation would cause no additional elevation in called cgs2 (DeVoti et al. 19911, which encodes cAMP the cAMP level in the pdel-disrupted cells. Alterna- phosphodiesterase. Disruption of pdel resulted in eleva- tively, if Gpa2 were involved in the regulation of cAMP tion of the intracellular cAMP level (Mochizuki and Ya- production, further elevation might have occurred. Anal-

Figure 5. Morphology of homothallic cells carrying mutant Gpa2 with low GTPase activity. The following plasmids were introduced into JY450 (h 9~ wild type), and the cells were incubated on SSA plates at 30~ for 5 days to induce mating and sporulation. pART3 is the vector, as a control, pAGWT carries the wild-type allele of gpa2. pAGH and pAGL carry mutant alleles R176H and Q202L, respectively. Construction of these mutant alleles is described in Materials and methods. Phase-contrast micrographs of JY450 cells transformed with each plasmid are shown in A-D. (A) The vector pART3; (B) pAGWT (gpa2+); {C) pAGH (gpa2R176H); (D) pAGL (gpa2Q2~ Bar, 10 ~m.

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S. pombe G,~ monitoring nutrition

Table 2. Mating efficiency of gpa2 mutants plays an important role in the assessment of these con- Mating ditions. We presented direct evidence that Gpa2 is re- Strains gpa2 allele efficiency (%) quired for sensing glucose stimulation. Judging from the phenotypes of gpa2 mutants, it is also possible that Gpa2 JY450 gpa2 + 92.1 + 1.8 detects the availability of nitrogen. JZ692 gpa2 Rt76H 8.7 + 1.2 Despite the above conclusions, however, our study is JZ593 gpa2 Qz~ 10.2 + 1.9 still too preliminary to answer questions regarding how cells monitor nutritional conditions. For instance, one may ask which kind of molecule represents nutritional conditions, or whether such a molecule exists at all. If ysis of the double mutants showed that the latter is the so, does a single molecule represent the availability of case {Table 1, experiments 4 and 5). A remarkable in- both nitrogen and carbon? Is Gpa2 coupled to a receptor crease in the level of cAMP was observed in both that binds a ligand? Is the receptor membrane bound? We gpa2 R176H pdel- strains (JZ657, JZ658) and gpa2 Q2~ presently have no answer to these fundamental ques- pdel- strains (JZ659, JZ660). Furthermore, the cAMP tions. Further analysis of the Gpa2 pathway will help to level in gpa2- pdel - strains (Table 1, JZ655 and JZ656) solve these problems. was much lower than that of the parental pdel - strains It has been established that G proteins coupled to hor- (JZ666, JZ667) and was comparable to that of the wild mone receptors regulate adenylyl cyclase in mammalian type. They were competent in mating. These results in- cells {for review, see Gilman 1987). Our conclusion is dicate that Gpa2 does not regulate the pdeI-encoded that Gpa2 is likely to regulate adenylyl cyclase in fission phosphodiesterase. Thus, Gpa2 is most likely to be in- yeast. This analogy is not as simple as it may appear. S. volved in the regulation of cAMP production in S. pombe adenylyl cyclase (Yamawaki-Kataoka et al. 1989; pombe, although we cannot completely rule out the pos- Young et al. 1989) resembles S. cerevisiae adenylyl cy- sibility that S. pombe has another phosphodiesterase, yet clase (Kataoka et al. 1985), but these yeast enzymes have undetected, and that its activity is regulated by Gpa2. only limited homology with mammalian adenylyl cyclases and the two groups apparently differ in their Gpa2 is required for the response to glucose means of association with the plasma membrane (Kru- stimulation pinski et al. 1989; for review, see Tang and Gilman 1992). Furthermore, S. cerevisiae adenylyl cyclase is reg- It has been shown that stimulation of quiescent S. cere- ulated by Ras proteins (Toda et al. 1985), but the only visiae cells by glucose results in a transient increase in Ras homolog in S. pombe, encoded by rasl, does not the intracellular cAMP level [Nakafuku et al. 1988). To regulate adenylyl cyclase activity (Fukui et al. 1986). test whether Gpa2 is involved in this type of response in S. pombe, gpa2 +, gpa2-, and gpa2 R176tt cells were starved for both glucose and nitrogen and then stimulated by the addition of glucose to a final concentration of 2%. The amount of cAMP in the cell was assayed at intervals, and the results are given in Figure 6. The cAMP level in the gpa2 + cells reached a peak 4 min after -E-- 3, stimulation and then decreased and stayed around the value of 2.0 pmoles/mg of protein. The gpa2- cells, >'5 however, showed no peak, and the level increased grad- ually and reached only 1.1 pmole/mg of protein. The R176H mutant showed a sharp increase in the level of cAMP, which then stayed at -2.7 pmoles/mg of protein. 0 I These results indicate that Gpa2 has a role in the assessment of glucose availability. o~ 0 10 20 30 40 Discussion time(min) This study has shown that S. pombe has a second G homolog. The first homolog, encoded by gpal, was char- Figure 6. Glucose-stimulated cAMP formation in gpa2 mu- acterized previously and concluded to be coupled with tants. Cells of JY333 (h- wild type), 17-393 (h- gpa2::ura4+), the mating pheromone receptors (Obara et al. 1991 ). The and JZ693 (h- gpa2 Rt76rt) were grown in MM to the stationary phase and collected. They were resuspended in MM-C-N and second homolog, encoded by gpa2, is not closely related incubated for 6-7 hr. These cells were stimulated with glucose to the first, structurally or functionally. [final concentration, 2% ), and the cAMP level was measured at Analysis of gpa2 mutants suggested that its gene prod- short intervals. Only one sample was assayed at each point, but uct Gpa2 is likely to be involved in regulation of cAMP essentially the same pattern was reproduced in a separate ex- production. The level of cAMP in S. pombe cells varies, periment. {O)JY333 (gpa2 +); {A)JZ393 (gpa2::ura4 +); (m)JZ693 depending on nutritional conditions. Gpa2 apparently {gpa2R176H).

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Thus, S. pombe adenylyl cyclase appears to occupy a Table 3. S. pombe strains used in this study curious intermediate position in terms of regulation by Strain Genotype the GTP-binding protein. An interesting observation has been made in S. cere- JY333 h - ade6-M216 leu 1 visiae relative to the above point. In the budding yeast, JY450 h 9~ ade6-M216 leu 1 two genes have been shown to encode G~ homologs. The JY765 h +/h - ade6-M216/ade6-M210 leul/leul first homolog, encoded by GPAI/SCG1, is coupled to the ura4-D 18/ura4-D 18 mating pheromone receptors (Dietzel and Kurjan 1987; JZ393 h - ade6-M216 leul ura4-D18 gpa2::ura4 + JZ415 h 9~ ade6-M216 leul ura4-D18 gpa2::ura4 + Miyajima et al. 1987). The role of Gpa2, the second ho- JZ593 h 9~ ade6-M216 leul gpa2 Q2~ molog encoded by the GPA2 gene, is less clear, because JZ655 h 9~ ade6-M216 leul ura4-D18 pdel::ura4 § the disruption of GPA2 did not cause any obvious phe- gpa2::ura4 + notype (Nakafuku et al. 1988). Overexpression of GPA2 Z656 h9O ade6-M210 leul ura4-D18 pdel:ura4 + elevated the cAMP level and could suppress a tempera- gpa2::ura4 + ture-sensitive RAS2 mutation. Although this observa- IZ657 hg0 ade6-M216 leu 1 ura4-D 18 pdel ::ura4 + tion was interpreted in several ways by Nakafuku et al. gpa2 R176H (1988), our finding that S. pombe Gpa2 is likely to regu- FZ658 hgo ade6-M210 leu 1 ura4-D 18 pdel ::ura4 + gpa2 RI76H late adenylyl cyclase suggests that S. cerevisiae Gpa2 rZ659 hgO ade6-M216 leul ura4-D18 pdel::ura4 + may also be capable of activating adenylyl cyclase. The gpa2 Q2~ similarity observed between S. pornbe Gpa2 and S. cer- FZ660 hgo ade6-M210 leu 1 ura4-D 18 pdel ::ura4 + evisiae Gpa2 supports this notion. These considerations gpa2 Q202L lead to the following concept. Both Ras proteins and a G rZ666 h 90 ade6-M216 teu 1 ura4-D 18 pde I:: ura4 + protein regulate the activity of adenylyl cyclase in S. FZ667 h 90 ade6-M210 leul ura4-D 18 pdel ::ura4 + cerevisiae, but the contribution of the G protein is un- [Z692 h 90 ade6-M216 leul gpa2 R176" important under physiological conditions. In contrast, rZ693 h - ade6-M216 leuI gpa2 R176" the Ras protein is not involved in the regulation of adenylyl cyclase in fission yeast (Fukui et al. 1986) and a G protein has established itself as the major regulator of the enzyme. by Southern analysis (Southern 1979) under low stringency con- It has been proposed that GTP-binding proteins are ditions using D. discoideum G~I eDNA as a probe. The strin- unlikely to be involved in the regulation of adenylyl cy- gency conditions employed in this study are as follows. Hybrid- clase in S. pombe, based on the observation that it has ization was done in 6x SSC (0.9 M NaC1/0.09 M sodium citrate) been impossible to reproduce enzymatic activity in vitro containing 0.1% N-lauroylsarcosinate, 0.02% sodium lauryl in a GTP-dependent manner {Engelberg et al. 1990). Our sulfate, 3% blocking reagent (Boehringer Mannheim), and 20% formamide for 15 hr at either 42~ or 49~ Washing was carried findings urge more careful investigation of this point, out in 2x SSC (0.3 M NaC1/0.03 M sodium citrate) containing especially because it is now possible to use genetically 0.1% sodium lauryl sulfate. Washing for 5 rain was repeated engineered Gpa2 protein, with or without a mutation, to three times at room temperature, and washing for 30 rain was establish the in vitro assay system. repeated three times at 53~ Two positive clones were eventu- ally obtained, which turned out to have the same insert. Sub- clones of the insert were made in pUC119. Because the original Materials and methods insert was found to carry only part of the gpa2 gene, a 2.4-kb PstI Strains, media, genetic methods, and transformation fragment that carried the missing 3'-terminal region of gpa2 was of S. pombe cloned in pUC 119 using a 0.1-kb ScaI-EcoRl fragment (Fig. 1) as the probe. S. pombe strains used in this study are listed in Table 3. Media for routine cultures and general genetic methods are according to Gutz et al. (1974}. Cells subjected to the analysis of cAMP DNA sequence determination were grown in minimal liquid medium (MM) and its deriva- Nucleotide sequences were determined by the dideoxy chain- tives. MM is the same as EMM, described by Moreno et al. termination method [Sanger et al. 1977). DNA fragments to be (1990) except that the concentration of molybdic acid is 0.13 sequenced were cloned in either pUC119 or pBluescript (Strat- rag/liter. MM-N lacks NH4C1, and MM-C-N lacks glucose and agene), and subclones for sequencing were generated by progres- NH4C1. Mating and sporulation efficiency were examined on sive deletion with exonuclease III and S1 nuclease (Henikoff SSA plates (Egel and Egel-Mitani 1974). Transformation of S. 1984). Single-stranded DNA was used as the template for se- pornbe was described previously (Beach et al. 1982; Okazaki et quence determination. The region shown in Figure 2 has been al. 1990). sequenced in both directions at least once.

Cloning of gpa2 Disruption of gpa2 S. pombe genomir DNA was digested with EcoRI and electro- One-step gene disruption of gpa2 was done essentially as de- phoresed in a 0.9% agarose gel. DNA fragments of -8 kb in scribed IRothstein 1983; Fukui et al. 1986). A pUC119-based length were recovered from a piece of the gel by using a silica plasmid was constructed by ligating the following three frag- matrix (Geneclean II Kit, Biol01, Inc.) and cloned in KZAP ments: a 1.1-kb BamHI-PstI fragment carrying the immediate (Stratagene). One thousand recombinant phages were screened 5' upstream element to the gpa20RF, of which the PstI end was

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S. pombe Get monitoring nutrition blunted; a 1.8-kb S. pombe ura4 + cassette {Grimm et al., 1988) Beals, C.R., C.B. Wilson, and R.M. Perlmutter. 1987. A small with both ends blunted; and a HincII-PstI fragment (0.9 kb) multigene family encodes Gli) signal-transduction proteins. carrying the immediate 3' downstream element to the gpa2 Proc. Natl. Acad. Sci. 84: 7886--7890. ORF and being connected to the vector pUC 119 linearized with Boeke, J.D., F. Lacroute, and G.R. Fink. 1984. A positive selec- PstI and BamHI. Digestion of this plasmid with BamHI and PstI tion for mutants lacking orotidine-5'-phosphate decarboxyl- gave a 3.7-kb BamHI-PstI fragment that covered the gpa2 locus ase activity in yeast: 5-Fluoro-orotic acid resistance. Mol. & but in which the 1.5-kb PstI-HinclI fragment carrying the entire Gen. Genet. 197: 345-346. gpa20RF was replaced by ura4 (Fig. 1). An S. pombe diploid Dever, T.E., M.J. Glynias, and W.C. Merrick. 1987. GTP binding strain, JY765 (h +/h- ade6-M216/ade6-M210 leul/leul ura4- domain: Three consensus sequence elements with distinct D18/ura4-D18), was transformed with this fragment, and Ura § spacing. Proc. Natl. Acad. Sci. 84: 1814-1818. transformants were selected. Disruption of one allele of gpa2 DeVoti, J., G. Seydoux, D. Beach, and M. McLeod. 1991. Inter- was confirmed by Southern blotting in these transformants. action between ranI + protein kinase and cAMP dependent protein kinase as negative regulators of fission yeast meiosis. EMBO I. 10: 3759-3768. Construction of mutant gpa2 alleles Dietzel, C. and J. Kurjan. 1987. The yeast SCGI gene: A Get-like In vitro site-directed mutagenesis of gpa2 was performed as de- protein implicated in the a- and et-factor response pathway. scribed {Kunkel 1985). The synthetic oligonucleotide used for Cell 50: 1001-1010. construction of the R176H mutation was 5'-ACCCAGAGTAC- Dohlman, H.G., J, Thomer, M.G. Caron, and R.J. Lefkowitz. TATTGTGAGACCGGAGAATGTC-3', and that for Q202L 1991. Model systems for the study of seven-transmembrane- was 5'-CCTTCGTTCGGTTCTTAAGCCACCAACGTCGAA- segment receptors. Annu. Rev. Biochem. 60: 653--688. 3'. Nucleotides that were changed from the wild-type sequence Egel, R. 1973. Commitment to meiosis in fission yeast. Mol. & are underlined. The R176H mutation eliminated an XbaI rec- Gen. Genet. 121: 277-284. ognition site, whereas the Q202L mutation created an AflII rec- Egel, R. and M. Egel-Mitani. 1974. Premeiotic DNA synthesis in ognition site. Successful construction of the mutant alleles was fission yeast. Exp. Cell Res. 88: 127-134. confirmed by sequence analysis. These mutant alleles were ei- Engelberg, D., E. Poradosu, G. Simchen, and A. Levitzki. 1990. ther borne on a multicopy plasmid or integrated in the chromo- Adenylyl cyclase activity of the fission yeast Schizosaccha- some. For integration, a 3.4-kb BamHI-SalI (PstI) fragment car- romyces pombe is not regulated by guanyl nucleotides. FEBS rying the mutated allele was introduced into an h 9~ gpa2 dis- Lett. 261: 413-418. ruptant, JZ415, in which the gpa20RF was replaced by a ura4 + Fukui, Y., T. Kozasa, Y. Kaziro, T. Takeda, and M. Yamamoto. cassette. Substitution of the disrupted allele with the mutant 1986. Role of a ras homolog in the life cycle of Schizosac- allele was expected to result in uracil auxotrophy. Ura- trans- charomyces pombe. Cell 44: 329-336. formants were chosen by 5-FOA selection (Boeke et al. 1984; Gilman, A.G. 1987. G protein: Transducers of receptor-gener- Grimm et al. 1988), and successful substitution was confirmed ated signals. Annu. Rev. Biochem. 56: 615--649. in some of them by Southern blotting. Grimm, C., J. Kohli, J. Murray, and K. Maundrell. 1988. Genetic engineering of Schizosaccharomyces pombe: A system for gene disruption and replacement using the ura4 gene as a cAMP assay selectable marker. Mol. & Gen. Genet. 215: 81-86. Gutz, H., H. Heslot, U. Leupold, and N. Lopfieno. 1974. Preparation of samples for cAMP assay was described previ- Schizosaccharomyces pombe. In Handbook of genetics (ed. ously (Fukui et al. 1986; Mochizuki and Yamamoto 1992). We R.D. King), vol. I, pp. 395-446. Plenum Publishing, New used a cAMP assay kit (Amersham TRK.432) and followed the York. protocol provided by the supplier. Samples to be compared were Halliday, K. 1984. Regional homology in GTP binding proto- assayed simultaneously using a single batch of the kit; other- oncogene products and elongation factors. J. Cyclic Nucle- wise, the results were not sufficiently reproducible (Mochizuki otide Res. 9: 435---448. and Yamamoto 1992). The amount of protein was determined Henikoff, S. 1984. Unidirectional digestion with exonuclease III according to Lowry et al. (1951). creates targeted breakpoints for DNA sequencing. Gene 28:351-359. Kataoka, T., D. Broek, and M. Wigler. 1985. DNA sequence and Acknowledgments characterization of the S. cerevisiae gene encoding adenylate We thank Dr. Richard Firtel for providing the cDNA clones cyclase. Cell 43: 493-505. encoding D. discoideum Gal and Get2. We also thank Dr. David Kawamukai, M., K. Ferguson, M. Wigler, and D. Young. 1991. Hughes for reading the manuscript. This work was supported by Genetic and biochemical analysis of the adenylyl cyclase of Grants-in-Aid from the Ministry of Education, Science, and Schizosaccharomyces pombe. Cell Regul. 2: 155-164. Culture of Japan. Kaziro, Y., H. Itoh, T. Kozasa, M. Nakafuku, and T. Satoh. 1991. The publication costs of this article were defrayed in part by Structure and function of signal transducing GTP-binding payment of page charges. This article must therefore be hereby proteins. Annu. Rev. Biochem. 60: 349-400. marked "advertisement" in accordance with 18 USC section Kelly, M., J. Burke, M. Smith, A. Klar, and D. Beach. 1988. Four 1734 solely to indicate this fact. mating-type genes control sexual differentiation in the fission yeast. EMBO ]. 7: 1537-1547. Kunkel, T.A. 1985. Rapid and efficient site-specific mutagenesis References without phenotypic selection. Proc. Natl. Acad. Sci. 82: 488-492. Beach, D., M. Piper, and P. Nurse. 1982. Construction of a Krupinski, J., F. Coussen, H.A. Bakalyar, W.-J. Tang, P.G. Fein- Schizosaccharomyces pombe gene bank in a yeast bacterial stein, K. Orth, C. Slaughter, R.R. Reed, and A.G. Gilman. shuttle vector and its use to isolate genes by complementa- 1989. Adenylyl cyclase amino acid sequence: Possible chan- tion. Mol. & Gen. Genet. 187: 326--329. nel- or transporter-like structure. Science 244: 1558-1564.

GENES & DEVELOPMENT 2461 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press

Isshiki et ai.

Landis, C.A., S.B. Masters, A. Spada, A.M. Pace, H.R. Bourne, critical regulator of sexual development. Genes & Dev. and L. Vallar. 1989. GTPase inhibiting mutations activate 5: 1990-1999. the chain of Gs and stimulate adenylyl cyclase in human Tang, W.-J., and A.G. Gilman. 1992. Adenylyl cyclases. Cell pituitary tumours. Nature 340: 692-696. 70: 869-872. Leupold, U. 1970. Genetical methods for Schizosaccharomyces Toda, T., I. Uno, T. Ishikawa, S. Powers, T. Kataoka, D. Brock, pombe. Methods Cell Physiol. 4: 169-177. S. Cameron, J. Broach, K. Matsumoto, and M. Wiglet. 1985. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall. 1951. In yeast, RAS proteins are controlling elements of adenylate Protein measurement with a phenol reagent. J. Biol. Chem. cyclase. Cell 40: 27-36. 193: 265-275. Watanabe, Y., Y. Iino, K. Furuhata, C. Shimoda, and M. Ya- Lyons, J., C.A. Landis, G. Harsh, L. Vallar, K. Grunewald, H. mamoto 1988. The S. pombe mei2 gene encoding a crucial Feichtinger, Q.-Y. Duh, O.H. Clark, E. Kawasaki, H.R. molecule for commitment to meiosis is under the regulation Bourne, and F. McCormick. 1990. Two G protein oncogenes of cAMP. EMBO J. 7: 761-767. in human endocrine tumors. Science 249: 655-659. Yamawaki-Kataoka, Y., T. Tamaoki, H.-R. Choe, H. Tanaka, Maeda, T., N. Mochizuki, and M. Yamamoto. 1990. Adenylyl and T. Kataoka. 1989. Adenylate cyclase in yeast: A com- cyclase is dispensable for vegetative cell growth in the fis- parison of the genes from Schizosaccharomyces pombe and sion yeast Schizosaccharomyces pombe. Proc. Natl. Acad. Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. 86: 5693- Sci. 87: 7814-7818. 5697. Miyajima, I., M. Nakafuku, N. Nakayama, C.B. Brenner, A. Young, D., M. Riggs, J. Field, A. Vojtek, D. Brock, and M. Wigler. Miyajima, K. Kaibuchi, K. Arai, Y. Kaziro, and K. Matsu- 1989. The adenylyl cyclase gene from Schizosaccharomyces moto. 1987. GPA1, a haploid-specific essential gene, encodes pombe. Proc. Natl. Acad. Sci. 86: 7989-7993. a yeast homolog of mammalian G protein which may be involved in mating factor signal transduction. Cell 50:1011-1019. Mochizuki, N. and M. Yamamoto. 1992. Reduction in the intracellular cAMP level triggers initiation of sexual development in fission yeast. Mol. & Gen. Genet. 233: 17-24. Moreno, S., A. Klar, and P. Nurse. 1990. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194: 793-823. Nakafuku, M., T. Obara, K. Kaibuchi, I. Miyajima, A. Miyajima, H. Itoh, S. Nakamura, K. Arai, K. Matsumoto, and Y. Kaziro. 1988. Isolation of a second yeast Saccharomyces cerevisiae gene (GPA2) coding for guanine nucleotide-binding regula- tory protein: Studies on its structure and possible functions. Proc. Natl. Acad. Sci. 85: 1374-1378. Obara, T., M.Nakafuku, M.Yamamoto, and Y. Kaziro. 1991. Iso- lation and characterization of a gene encoding a G-protein a subunit from Schizosaccharomyces pombe: Involvement in mating and sporulation pathways. Proc. Natl. Acad. Sci. 88: 5877-5881. Okazaki, K., N. Okazaki, K. Kume, S. Jinno, K. Tanaka, and H. Okayama. 1990. High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of Schizosaccharomyces pombe. Nucleic Acids Res. 18: 6485-6489. Pupillo, M., A. Kumagai, G.S. Pitt, R.A. Firtel, and P.N. Devreotes. 1989. Multiple a subunits of guanine nucleotide- binding proteins in Dictyostelium. Proc. Natl. Acad. Sci. 86: 4892-4896. Rothstein, R. 1983. One step gene disruption in yeast. Methods Enzymol. 101: 202-211. Russell, P. 1989. Gene cloning and expression in fission yeast. In Molecular biology of the fission yeast (ed. A. Nasim et al.J, pp. 243-271. Academic Press, New York. Saiki, R.K., D.H. Gelfand, S. Stoffel, S.J. Scharf, R. Higuchi, G.T. Horn, K.B. Mullis, and H.A. Erlich. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491. Sanger, F., S. Nicklen, and A.R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. 74: 5463-5467. Southern, E. 1979. Gel electrophoresis of restriction fragments. Methods Enzymol. 68: 152-176. Sugimoto, A., Y. Iino, T. Maeda, Y. Watanabe, and M. Ya- mamoto. 1991. Schizosaccharomyces pombe ste11 + encodes a transcription factor with an HMG motif that is a

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Characterization of a fission yeast gene, gpa2, that encodes a G alpha subunit involved in the monitoring of nutrition.

T Isshiki, N Mochizuki, T Maeda, et al.

Genes Dev. 1992, 6: Access the most recent version at doi:10.1101/gad.6.12b.2455

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